Lipases (triacyl glycerol hydrolase, EC 3.1.1.3) are widely distributed in nature. The principal biological function of lipase is the breakdown of lipids as an initial event in the utilization of fat as an energy source. The characteristic properties such as substrate specificity, regioselectivity and enantioselectivity among various lipases allow wide applications such as in the production of emulsifiers, fatty acid esters, fatty acids, and carbohydrate derivatives. (Liu & Shaw, J. Am. Oil. Chem. Soc. (1995), 72:1271-1274; Shaw & Wang, Enzyme Microb. Technol. (1991), 13:544–546; Wang et al., Biotechnol. Bioeng. (1992), 39:1128–1132 (1992)). A Ser-His-Asp catalytic triad occurs in lipases, which are responsible for hydrolyzing triglycerides into diglycerides and subsequently, monoglycerides and free fatty acids (Wallace et al., Protein Science (1996), 5:1001–1013).
The production of lipases is a general property of staphylococci, and lipase genes have been identified in Staphylococcus aureus strains PS54 and NCT8530, Staphylococcus epidermidis strain 9 and Staphylococcus hyicus. (Simons et al., Eur. J. Biochem. (1998), 253:675–683). The family of lipases from staphylococci demonstrates common structural features. For example, these enzymes are produced as preproenzymes, which have molecular masses of approximately 70 kDa. After secretion into the growth medium, proteolytic processing results in mature forms with molecular masses of 40–46 kDa. (Nikoleit et al., Eur. J. Biochem. (1995), 228:732–738).
Staphylococcus epidermidis strain 9 lipase gene (gehC) consists of a single open reading frame of 2064 nucleotides, which encoded a protein of 688 amino acids with a predicted molecular mass of 77 kDa. The gehC gene has been cloned and expressed in Escherichia coli by Simons et al (Eur. J. Biochem. 1998: 253(3): 675–683). In the extracts of E. coli harboring gehC, a lipase corresponding to the 77-kDa lipase but with an electrophoretic mobility equivalent to a 97-kDa protein has been detected. In the supernatant fluid of S. epidermidis strain 9 culture, a 43-kDa lipase of 386 amino acids has also been identified. It was suggested that the S. epidermidis lipase of 97 kDa electrophoretic mobility was secreted as a proprotein and subsequently cleaved between the Ala-302 and Lys-303 mino acid residues by a proteolytic enzyme to yield the 43-kDa lipase. However, no further structure-function studies on the Ser-His-Asp triad of the 43-kDa lipase have been carried out.
The lipase gene gehSE1 isolated from S. epidermidis strain RP62A was organized as a preproenzyme. A part of gehSE1 gene encoded the mature lipase of 380 amino acids that had a sequence similar to that of S. epidermidis strain 9 lipase from Asn-7 to Lys-386 with 97.8% homology. The gehSE1 mature lipase has been overexpressed as a fusion protein with an N-terminal His-tag in E. coli and characterized by Farrell et al., J. Gen. Microbiol. (1993), 139:267–277.
Lipases have become increasingly important in biotechnology. The characteristic properties such as substrate specificity, regioselectivity and enantioselectivity among various lipases allow their wide applications, such as in the productions of emulsifiers, fatty acid esters, fatty acids and carbohydrate derivatives. In addition, there has been a strong demand in recent years for natural products, including natural flavors.
Esters are common flavor agents and are often employed in fruit-flavored products (e.g., beverages, candies, jellies, and jams), baked goods, wines, and dairy products (e.g., cultured butter, sour cream, yogurt, and cheese). Naturally occurring esters have been isolated from all major food systems and often are expensive.
Conventional production of flavor esters using chemical-catalyzed esterification requires high temperature and leads to dark-colored products and undesired byproducts. Enzyme-catalyzed conversion (biocatalysis) provides an alternative to the chemical syntheses of flavor esters. It is more efficient and selective. Inexpensive natural raw materials, such as fatty acids and alcohols, can be used in the enzyme-catalyzed synthesis of flavor esters (Manjon et al., Biotechnol. Lett. (1991), 13:339–344).
The use of lipolytic enzymes to catalyze the esterification reaction for producing flavor esters has been investigated by many workers. However, current methods using lipolytic enzymes were performed in hydrophobic organic solvents or in aqueous-organic two-phase systems. The uses of organic solvents carry the risks of flammability as well as toxicity to the production workers and the environment. Residual organic solvents may cause a safety concern for the consumers. Thus, the needs for a safe and more effective biocatalytic system remain unfulfilled.